Directed differentiation of human pluripotent stem cells into functional insulin-producing beta-like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non-functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1 + and subsequent PDX1 + /NKX6.1 + pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1 + /NKX6.1 + progenitors produces glucose-responsive beta-like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short-term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta-like cells.
Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional β cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and β cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature β-like cells to form islet-sized enriched β-clusters (eBCs). eBCs display physiological properties analogous to primary human β cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell *
The mammalian Golgi apparatus exists as stacks of cisternae that are laterally linked to form a continuous membrane ribbon, but neither the molecular requirements for, nor the purpose of, Golgi ribbon formation are known. Here, we demonstrate that ribbon formation is mediated by specific membrane-fusion events that occur during Golgi assembly, and require the Golgi proteins GM130 and GRASP65. Furthermore, these GM130 and GRASP65-dependent lateral cisternal-fusion reactions are necessary to achieve uniform distribution of enzymes in the Golgi ribbon. The membrane continuity created by ribbon formation facilitates optimal processing conditions in the biosynthetic pathway.
Peri-centrosomal positioning of the mammalian Golgi apparatus is known to involve microtubule-based motility, but its importance for cellular physiology is a major unanswered question. Here, we identify golgin-160 and GMAP210 as proteins required for centripetal motility of Golgi membranes. In the absence of either golgin, peri-centrosomal positioning of the Golgi apparatus was disrupted while the cytoskeleton remained intact. Although secretion persisted with normal kinetics, it was evenly distributed in response to wounding rather than directed to the wound edge. Strikingly, these cells also completely failed to polarize. Further, directionally persistent cell migration was inhibited such that wound closure was impaired. These findings not only reveal novel roles for golgin-160 and GMAP210 in conferring membrane motility but also indicate that Golgi positioning has an active role in directed secretion, cell polarity, and wound healing.
Pancreatic β cells are highly specialized to regulate systemic glucose levels by secreting insulin. In adults, increase in β-cell mass is limited due to brakes on cell replication. In contrast, proliferation is robust in neonatal β cells that are functionally immature as defined by a lower set point for glucose-stimulated insulin secretion. Here we show that β-cell proliferation and immaturity are linked by tuning expression of physiologically relevant, non-oncogenic levels of c-Myc. Adult β cells induced to replicate adopt gene expression and metabolic profiles resembling those of immature neonatal β that proliferate readily. We directly demonstrate that priming insulin-producing cells to enter the cell cycle promotes a functionally immature phenotype. We suggest that there exists a balance between mature functionality and the ability to expand, as the phenotypic state of the β cell reverts to a less functional one in response to proliferative cues.
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